House dust mites (HDM) are a common source of allergens worldwide. Dermatophagoïdes pteronyssinus is one of the prevalent mite species in Europe and North America. Der p 2 is one of the major allergens of D. pteronyssinus. This protein is highly clinically relevant since a majority of mite allergic patients exhibit high seric IgE titres against this molecule (Pittner et al., 2004; Thomas et al., 2002; Weghofer et al., 2008).
One of the best therapeutic options for patients suffering from allergies is specific allergy vaccination which, in most cases, reduces or alleviates the allergic symptoms caused by the allergen in question. Conventional specific allergy vaccination is a causal treatment for allergic disease. It interferes with basic immunological mechanisms resulting in persistent improvement of the patients' immune status. Thus, the protective effect of specific allergy vaccination extends beyond the treatment period in contrast to symptomatic drug treatment. Some patients receiving the treatment are cured, and, in addition, most patients experience a relief in disease severity and symptoms experienced, or at least an arrest in disease aggravation.
Given that they are well characterised at the molecular level, recombinant allergens are an alternative to the complex biological extracts used in specific allergy vaccination. Their biological properties such as immunogenicity and safety depend on the chosen expression system.
When using the sublingual route for immunotherapy, protein in a natural conformation and preserved immunogenicity in terms of IgE, IgG, and T lymphocyte recognition are preferred (Moingeon et al., 2006). Also, an allergen in a natural-like conformation has been successfully used for desensitization purposes in humans (Pauli et al., J. Allergy Clin Immunol 2008, 122(5) 951-960). Such “natural-like” molecules are further needed for diagnostic purposes.
To replace biological extracts, there is a need for a recombinant house dust mite allergen with a high purity, a natural conformation, and conserved antigenicity and immunogenicity (both in terms of IgE, IgG, and T lymphocyte recognition) as compared with the natural allergen.
Der p 2 is a 14.1 kDa protein comprising 3 disulfide bonds. When recombinantly expressed in E. coli, the protein forms insoluble inclusion bodies and thus must be refolded after solubilisation with denaturing agents (Takai et al., 2005). Besides, although the recombinant protein thus obtained seems to contain the 3 correct disulfide bonds, it also displays a series of unexpected disulfide bonds (Table 1 of Example 2 below). Lastly, recombinant Der p 2 expressed in E. coli shows less direct IgE binding in a serum than natural Der p 2 (Takai et al., 2005).
Refolding the allergen out of inclusion bodies to obtain a natural conformation can be difficult, especially with proteins comprising multiple disulfide bonds. Yeasts can thus be considered as alternative expression hosts.
Expression of Der p 2 in the budding yeast S. cerevisiae has been described. It yielded a molecule with a preserved immunogenicity when compared to the natural protein (Hakkaart et al., 1998). However, conserved immunoreactivity and immunogenicity have been observed for Der p 2 allergen despite incorrect folding (Bussières et al., 2010). Thus a detailed structural characterisation, in particular with respect with cysteine pairings, is needed to confirm the natural conformation of the protein.
Expression of Der p 2 in the P. pastoris yeast has been described (Tanyaratsrisakul et al., 2009). However, the recombinant protein produced by Tanyaratsrisakul et al. displayed a primarily random structure and needed refolding by precipitation/renaturation. It was shown that this refolded recombinant protein contained disulfide bonds, but not that these bonds implied the same cysteine residues as in the native Der p 2 protein. Furthermore, the protein obtained by Tanyaratsrisakul et al. differed from the native Der p 2 as it lacked an hydrophobic cavity. Lastly, immunoreactivity of this protein was only tested by IgE ELISA inhibition, which may show reactivity even in the case of denatured Der p 2 proteins as Der p 2 contains linear epitopes.
In conclusion, previous attempts to produce a recombinant Der p 2 protein similar to its natural counterpart had failed, yielding at best a molecule with partial folding.
Despite the previous unsuccessful attempt of Tanyaratsrisakul et al., the inventors have shown that it is possible to produce in P. pastoris a recombinant Der p 2 protein that spontaneously folds into a secondary structure similar to that of the native protein. The recombinant protein thus produced also contains disulfide bonds similar to that of the native protein, and is well-recognised by IgE as well as T-lymphocytes.